The ability of an anti-viral pathogen vaccine to produce an effective antibody response is typically evaluated in various types of virus neutralization tests or assays. Common features of such tests include monitoring the level of infectivity of the virus (natural or attenuated) in a standardized target cell culture, and evaluating the reduction in infectivity of the virus after incubation with the tested serum/sera or antibody solution(s) of interest.
The dilution of a serum or antibody (Ab) solution that provides 50% or more reduction of infectivity is referred to as the ‘neutralization titer’ (Niedrig et al. (2008) Clin. Vaccine Immunol. 15, 177; Guidelines for Plaque-Reduction Neutralization Testing of Human Antibodies to Dengue Viruses, WHO, 2007). Virus neutralization assays are widely used worldwide for a variety of viruses, from the relatively common, such as influenza and herpes simplex, to the most feared and dangerous viruses, such as smallpox, yellow fever, and dengue hemorrhagic fever (Niedrig et al. (2008) Clin. Vaccine Immunol. 15, 177; Guidelines for Plaque-Reduction Neutralization Testing of Human Antibodies to Dengue Viruses, WHO, 2007).
In the virology community, the plaque reduction neutralization test (PRNT) is generally considered the gold standard of neutralization assays for studying anti-viral humoral immune responses (Niedrig et al. (2008) Clin. Vaccine Immunol. 15, 177; Guidelines for Plaque-Reduction Neutralization Testing of Human Antibodies to Dengue Viruses, WHO, 2007; Roukens et al. (2008) PLoS ONE, 3, e1993; Niedrig et al. (1999) Trop. Med. Int. Health 4, 867). In the PRNT, a highly diluted virus culture, with or without various concentrations of a test serum or an antibody solution, is added to a culture of a confluent layer of target cells, and the level of infectivity is measured by the number of cell-free lacunas appearing in the culture after a period of incubation (typically a few days).
Currently, various more recent immunosorption methods compete with the PRNT, such as the immunofluorescent assay (IFA) (Kraus et al. (2007), J. Clin. Microbiol. 45, 3777; Niedrig et al. (1999) Trop. Med. Int. Health 4, 867; Groot & Riberiro (1962) Bull. WHO 27, 699; Vazquez et al. (2003) J. Virol. Methods 110, 179; Barry et al. (1991) Am. J. Trop. Med. Hyg. 44, 79; Deubel et al. (1983) Am. J. Trop. Med. Hyg. 32, 565)), where the infectivity level of virus is evaluated using fluorescently-labeled, virus-specific antibodies applied to fixed samples of target cells after incubation with live virus and washing. The IFA, although sensitive and virus-specific, remains under consideration, because the antibody titers from the IFA often do not correlate well with those from the PRNT (Niedrig et al. (1999) Trop. Med. Int. Health 4, 867; Groot & Riberiro (1962) Bull. WHO 27, 699; Vazquez et al. (2003) J. Virol. Methods 110, 179; Barry et al. (1991) Am. J. Trop. Med. Hyg. 44, 79; Deubel et al. (1983) Am. J. Trop. Med. Hyg. 32, 565).
A microneutralization method similar to the IFA, but using enzyme-linked immunosorption, was developed in Centers for Disease Control and Prevention (CDC). In this assay, the level of virus infectivity with or without tested sera is estimated by measuring nuclear protein (NP) of the avian influenza virus expressed in the target MDCK cells by staining the permeabilized fixed cells with the NP-specific monoclonal Ab labeled with horseradish peroxidase (HRP) (Rowe et al. (1999) J. Clin. Microbiol. 37, 937-943).
Another direct microneutralization (MN) assay is also practiced by the World Health Organization (WHO). In the MN assay, a confluent layer of target cells is infected with a diluted virus culture either in the presence or absence of a test serum/sera or antibody solution(s) of interest, and the rate of virus reproduction is evaluated by measuring released virus concentrations with a standard hemagglutination assay (HA) technique. The assay is described in the “WHO Manual on Animal Influenza Diagnosis and Surveillance,” (WHO/CDS/CSR/NCS/2002.5 Rev. 1) and may be found at the website beginning with “www.” and ending with “who.int/vaccine_research/diseases/influenza/WHO_manual_on_animal-diagnosis_and_surveillance—2002—5.pdf”. The protocol is simple and straightforward, although use of the HA technique presents certain limitations to the sensitivity of the method.
In general, the PRNT, IFA and MN have several complicating features:                Use of live virus raises safety and personnel protection issues.        Incubation of the target cells with live virus often requires significant time (up to ˜5-7 days) to allow for infection to properly develop and infectivity to be reliably evaluated, making the tests lengthy and cumbersome.        PRNT and IFA include multi-step fixation and staining protocols.        
The use of modern flow cytometry techniques promises to significantly improve existing neutralization assays. In several published studies, viral infection of target cells in the presence or absence of a test serum/sera or antibody solution(s) of interest has been monitored by flow cytometric methods, where viruses or their cell-expressed protein components were stained with fluorescent tags (Kremser et al. (2004) Anal. Chem. 80, 7360; Sliva et al. (2004) Virol. J. 1, 14; You et al. (2006) Int. J. Nanomedicine 1, 59; Nichols et al. (1993) Arch. Virol. 130, 441; Klingen et al. (2008) J. Virol. 82, 237; Lonsdale et al. (2003) J. Virol. Meth. 110, 67-71; Wang et al. (2004) J. Virol. Meth. 120, 207-215; Collins & Buchholz (2005) J. Virol. Meth. 128, 192-197). For example, permeabilized target cells were stained with fluorescent antibodies specific to target-expressed viral proteins (Lonsdale et al. (2003) J. Virol. Meth. 110, 67-71), and green fluorescent protein (GFP) was incorporated in a recombinant vector and expressed in infected cells (You et al. (2006) Int. J. Nanomedicine 1, 59; Wang et al. (2004) J. Virol. Meth. 120, 207; Collins & Buchholz (2005) J. Virol. Meth. 128, 192; Earl et al. (2003), J. Virol. 77, 10684). Other examples include direct labeling of the virus (Kremser et al. (2004) Anal. Chem. 80, 7360; Klingen et al. (2008) J. Virol. 82, 237). Such labeling can be performed via direct chemical tagging with fluorochromes (Kremser et al. (2004) Anal. Chem. 80, 7360), or electrostatic attachment of quantum dots to the envelope proteins of the virus (Sliva et al. (2004) Virol. J. 1, 14). Nichols et al. (1993; Arch. Virol. 130, 441) and Klingen et al. (2008; J. Virol. 82, 237) developed an ingenious virus staining method, growing the virus on a pre-stained target cells.
These flow cytometric methods, however, also use live viruses or recombinant vectors, raising safety and other issues similar to those listed for PRNT and IFA assays. These methods, while sensitive and informative in the research setting, can hardly be considered appropriate as routine high-throughput assays. Thus, there is a continuing need for rugged, reliable, and sensitive laboratory methods for microneutralization assays.